1. Technical Field
The present invention pertains to the production of phytase in transgenic plants and the use of the thus-produced phytase in industrial processes.
2. Background of the Invention
Phosphorus is an essential element for the growth of all organisms. In livestock production, feed must be supplemented with inorganic phosphorus in order to obtain a good growth performance of monogastric animals (e.g. pigs, poultry and fish).
In contrast, no inorganic phosphate needs to be added to the feedstuffs of ruminant animals. Microorganisms, present in the rumen, produce enzymes which catalyze the conversion of phytate (myo-inositolhexakis-phosphate) to inositol and inorganic phosphate.
Phytate occurs as a storage phosphorus source in virtually all feed substances originating from plants (for a review see: Phytic acid, chemistry and applications, E. Graf (ed.), Pilatus Press; Minneapolis, Minn., U.S.A. (1986)). Phytate comprises 1-3% of all nuts, cereals, legumes, oil seeds, spores and pollen. Complex salts of phytic acid are termed phytin. Phytic acid is considered to be an anti-nutritional factor since it chelates minerals such as calcium, zinc, magnesium, iron and may also react with proteins, thereby decreasing the bioavailability of proteins and nutritionally important minerals.
Phytate phosphorus passes through the gastrointestinal tract of monogastric animals and is excreted in the manure. Though some hydrolysis of phytate does occur in the colon, the thus-released inorganic phosphorus has no nutritional value since inorganic phosphorus is absorbed only in the small intestine. As a consequence, a significant amount of the nutritionally important phosphorus is not used by monogastric animals, despite its presence in the feed.
The excretion of phytate phosphorus in manure has further consequences. Intensive livestock production has increased enormously during the past decades. Consequently, the amount of manure produced has increased correspondingly and has caused environmental problems in various parts of the world. This is due, in part, to the accumulation of phosphate from manure in surface waters which has caused eutrophication.
The enzymes produced by microorganisms, which catalyze the conversion of phytate to inositol and inorganic phosphorus are broadly known as phytases. Phytase producing microorganisms comprise bacteria such as Bacillus subtilis (V. K. Paver and V. J. Jagannathan (1982) J. Bacteriol. 151, 1102) and Pseudomonas (D. J. Cosgrove (1970) Austral. J. Biol. Sci. 23, 1207); yeasts such as Saccharomvces cerevisiae (N. R. Nayini and P. Markakis (1984) Lebensmittel Wissenschaft und Technologie 17, 24); and fungi such as Aspergillus terreus (K. Yamada, Y. Minoda and S. Yamamoto (1986) Agric. Biol. Chem. 32, 1275). Various other Aspergillus species are known to produce phytase, of which, the phytase produced by Aspergillus ficuum has been determined to possess one of the highest levels of specific activity, as well as having better thermostability than phytases produced by other microorganisms (van Gorcom et al. (1991) European Patent Application 89202436.5, Publication No. 0 420 358, filed Sep. 27, 1989).
Phytases are also endogenously present in many plant species (see Loewus, F. A. (1990) In: Plant Biology vol. 9: "Inositol metabolism in plants" (eds. D. J. Morre, W. F. Boss, F. A. Loewus) 13). Gellatly, K. S. and Lefebvre, D. D. ((1990) Plant Physiology (supplement), 93, abstract 562) mention the isolation and characterization of a phytase cDNA clone obtained from potato tubers. Gibson, D. M. e al. and Christen, A. A. et al. ((1988) J. Cell Biochem., 12C abstracts L407 and L402, respectively) mention the synthesis of endogenous phytase during the germination of soybean seeds. However, plant phytases are normally produced in amounts insufficient for their application in industrial processes, per se.
The concept of adding microbial phytase to the feedstuffs of monogastric animals has been previously described (Ware, J. H., Bluff, L. and Shieh, T. R. (1967) U.S. Pat. No. 3,297,548; Nelson, T. S., Shieh, T. R., Wodzinski, R. J. and Ware, J. H. (1971) J. Nutrition 101, 1289). To date, however, application of this concept has not been commercially feasible, due to the high cost of the production of the microbial enzymes (Y. W. Han (1989) Animal Feed Sci. and Technol. 24, 345). For economic reasons, inorganic phosphorus is still added to monogastric animal feedstuffs.
Phytases have found other industrial uses as well. Exemplary of such utilities is an industrial process for the production of starch from cereals such as corn and wheat. Waste products comprising e.g. corn gluten feeds from such a wet milling process are sold as animal feed. During the steeping process phytase may be supplemented. Conditions (T.apprxeq.50.degree. C. and pH=5.5) are ideal for fungal phytases (see e.g. European Patent Application 0 321 004 to Alko Ltd.). Advantageously, animal feeds derived from the waste products of this process will contain phosphate instead of phytate.
It has also been conceived that phytases may be used in soy processing (see Finase.TM. Enzymes By Alko, a product information brochure published by Alko Ltd., Rajamaki, Finland). Soybean meal contains high levels of the anti-nutritional factor phytate which renders this protein source unsuitable for application in baby food and feed for fish, calves and other non-ruminants. Enzymatic upgrading of this valuable protein source improves the nutritional and commercial value of this material.
The possibility of using transgenic plants as a production system for valuable proteins has been proposed. Examples to date are the production of interferon in tobacco (Goodman, R. M., Knauf, V. C., Houck, C. M. and Comai, L. (1987) PCT/WO 87/00865), enkephalins in tobacco, Brassica napus and Arabidopsis thaliana (Vandekerckhove, J., Van Damme, J., Van Lijsebettens, M., Botterman, J., DeBlock, M., DeClerq, A., Leemans, J., Van Montagu, M. and Krebbers, E. (1989) Bio/Technol. 7, 929), antibodies in tobacco (Hiatt, A., Cafferkey, R. and Boedish, K. (1990) Nature 342, 76) and human serum albumin in tobacco and potato (Sijmons, P. C., Dekker, B. M. M., Schrammeijer, B., Verwoerd, T. C., van den Elzen, P. J. N. and Hoekema, A. (1990) Bio/Technol. 8, 217).
In practice, the transformation of an increasing number of plant species, especially dicotyledonous species (e.g. tobacco, potato, tomato, Petunia, Brassica), has become a routine procedure for workers skilled in the art (Klee, H., Horsch, R. and Rogers, S. (1987) Annu. Rev. Plant Physiol. 38, 467; Gasser C. S. and Fraley, R. T. (1989) Science 244, 1293). Strategies for the expression of foreign genes in plants have become well established (Gasser and Fraley, supra). Regulatory sequences from plant genes have been identified that are used for the construction of chimeric genes that can be functionally expressed in plants and plant cells.
For the introduction of gene constructions into plants, several technologies are available, such as transformation with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Using this strategy, a wide variety of plant tissues have been exploited, the choice being largely dependent on the plant species and its amenability in tissue culture. Successful examples are the transformation of protoplasts, microspores or pollen, and explants such as leaves, stems, roots, hypocotyls and cotyls. Furthermore, methods for direct DNA introduction in protoplasts and plant cells or tissues are used such as microinjection, electroporation, particle bombardment and direct DNA uptake (Gasser and Fraley, supra).
Proteins may be produced in plants using a variety of expression systems. For instance, the use of a constitutive promoter such as the 35S promoter of Cauliflower Mosaic Virus (CaMV) (Guilley, H., Dudley, R. K., Jonard, G., Balazs, E. and Richards, K. E. (1982) Cell 30, 763) will result in the accumulation of the expressed protein in all organs of the transgenic plant. Alternatively, use may be made of promoters from genes encoding proteins which are expressed in a highly tissue-specific and stage-specific manner (Higgins, T. J. V., (1984) Annu. Rev. Plant Physiol. 35, 191; Shotwell, M. A. and Larkins, B. A. (1989) In: The biochemistry of plants Vol. 15 (Academic Press, San Diego: Stumpf, P. K. and Conn, E. E., eds.), 297), i.e., the genes are expressed only in the target tissue and only during the desired stage of development.
It will be appreciated that an economical procedure for the production of phytase will be of significant benefit to, inter alia, the animal feed industry. One method of producing a more economical phytase would be to use recombinant DNA techniques to produce transgenic plants or plant organs capable of expressing phytase which could then in turn be added as such, for example, to animal food or feedstuffs for direct consumption by the animal. Alternatively, the phytase expressed in these transgenic plants or plant organs could be extracted and if desired, purified for the desired application.